Estrogen plays a critical role in female reproduction. Within the ovary, estrogen acts together with FSH and a variety of paracrine factors, such as IGF-1 and activin, to induce ovarian follicle maturation. Estrogen action is mediated through nuclear estrogen receptors (ER) alpha and beta, which act by altering gene expression and by modifying signaling pathways. ERalpha is expressed primarily in ovarian theca and stroma cells whereas ERbeta is predominant in granulosa cells. The estrogen receptor modulates gene transcription by two fundamentally different mechanisms: (1) via a classical estrogen response element (ERE)-mediated pathway and (2) via a non-classical or tethered pathway that involves ER interactions with other transcription factors present on target genes. We have created a novel knock-in mouse model in which two amino acids in the DNA-binding domain of ERalpha were mutated to Ala (ER[AA]), thereby selectively eliminating classical pathway signaling. Heterozygous females containing one mutant ER allele and one wild-type ER allele (ER AA/WT) are infertile-- they are anovulatory, exhibit increased follicular atresia, and have increased numbers of lipid-laden stromal cells. We hypothesize that the phenotypic features of this mouse model reflect the selective loss of gene expression via the classical ERE-mediated pathway. This unique genetic model will be crossed to ER knockout mice (ER -/-) to examine the distinct roles of the ERalpha classical and nonclassical pathways in ovarian function. Aim 1 will examine gene dosage effects of the ER AA mutation on follicular development and ovulation in the following six ERalpha genotypes: ER WT/WT, ER AA/WT, ER AA/AA, ER WT/-, ER AA/-, and ER -/-. Aim 2 will use microarrays in combination with analyses of genes known to regulate ovarian function to characterize genes regulated by the classical and nonclassical pathways. Aim 3 will examine the mechanism by which the ER AA mutant alters cellular function by using a combination of in vitro transfection studies and analyses of gene regulation in cells derived from the various ER genotypes. Aim 4 will examine how the ER AA mutation alters ER-mediated neuroendocrine signaling and gonadotropin stimulation of the ovary. Taken together, these proposed studies will use the ER AA genetic model to gain insight into how estrogen acts by classical and nonclassical pathways to regulate ovarian function.